The Na,K-ATPase comprises a group of isozymes responsible for maintaining the Na+ and K+ gradients across the plasma membrane of most cells. Isozyme diversity for the Na,K-ATPase results from the association of different molecular forms of the catalytic a and glycosylated p subunits that constitute the enzyme. Among these isozymes, a distinctive Na,K-ATPase composed of the oc4 isoform is selectively expressed in testis, where it is restricted to male germ cells. We have shown that the a4 isoform exhibits enzymatic properties that are unique, which suggested that the polypeptide plays a role in sustaining the ion gradients, membrane potential and excitability of male germ cells. In addition, we have found that a4 undergoes significant developmental changes, increasing dramatically after meiosis of the gametes. Most importantly, inhibition of a4 impairs sperm motility. These findings support an important role for a4 in sperm physiology and show that the protein is an attractive target for contraception. The Na,K-ATPase is the only known receptor for cardenolides, a group of compounds that are steroidal in nature. Our laboratory and others have reported that the cardenolide ouabain differentially inhibits the transport and catalytic activity of the Na,K-ATPase isozymes, being a4 the isoform that presents the highest sensitivity for ouabain. At present, the effect of cardenolides different from ouabain on a4 is unknown. Finding compounds that have a more restricted effect and preferentially inhibit a4 will be important to specifically interfere with Na+ and K+ transport in sperm. Our hypothesis is that certain cardenolides preferentially inhibit a4 over the other Na,K-ATPase isoforms, and that cardenolides and compounds which interfere with a.4 activity can affect sperm function and thus, can be used as male contraceptives. Specifically, the aims of this proposal are: 1) to identify cardenolides and via high throughput screening (HTS) compounds that affect activity of Na,K-ATPase a4 isoform, 2) to establish the effect of cardenolides and other a4 inhibitors on the catalytic and transport activity of the oc4 isoform from spermatozoa, 3) to determine the effect of cardenolides and compounds that inhibit a4 activity on sperm physiology, and 4) to identify the mechanisms by which inhibitors of the a4 isoform affect sperm function. This study will provide a new pharmacological approach to male contraception, and because cardenolides have a reversible effect, they will be useful for the temporary control of male fertility.